Biomedical subjects

R J Ruch

Publications and source records attributed to R J Ruch.

At least 37 records · Page 2Linked to original sources

PubMed1996

Inhibition of lung tumor cell growth in vitro and mouse lung tumor formation by lovastatin.

The HMG-CoA reductase inhibitor, lovastatin (LOV), has been reported to inhibit Ras farnesylation and the growth of Ras-transformed cells. Mouse lung tumors and human lung adenocarcinomas often have activating mutations in K-ras alleles. In the present study, we determined whether LOV inhibited the growth in vitro of mouse (C10, E9, LM1, LM2, and 82-132) and human (NCl-H125, H292, H441, H460, and H661) nor-transformed and neoplastically transformed lung epithelial cells and whether growth inhibition was related to cell transformation or K-ras activation. LOV inhibited the growth of mouse and human lung cells, but cell sensitivities were unrelated to neoplastic transformation or K-ras mutation. In addition, we evaluated whether LOV could inhibit the formation of lung adenomas induced by the tobacco-specific nitrosamine, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mice. LOV was administered in the diet at 0, 40, 160, or 400 ppm ad libitum to male strain A/J mice beginning 1 week after lung tumor induction with NNK (10 mumol/mouse). Mice were euthanized 6 months later. Enumeration of lung tumors revealed that LOV did not affect tumor incidence or size, but significantly reduced tumor multiplicity in a dose-related manner. These data suggest that LOV can suppress the formation of NNK-induced lung tumors, possibly at an early promotional stage. This suppression does not appear to be related to either the presence of mutated K-ras or to changes in K-ras expression.

Animals↗

PubMed1996

Lack of growth inhibition or enhancement of gap junctional intercellular communication and connexin43 expression by beta-carotene in murine lung epithelial cells in vitro.

The carotenoid, beta-carotene, has been examined in human trials as a possible lung cancer chemopreventive agent, but initial results indicate that the compound is ineffective. Here we have considered whether beta-carotene could enhance gap junctional intercellular communication (GJIC) and affect the growth of lung epithelial cells, since these effects may be involved in the carotenoid's chemopreventive actions. In accordance with its lack of lung cancer chemopreventive activity, beta-carotene (1-10 microM; 1-5 days treatment durations) did not affect GJIC, gap junction protein (connexin43; Cx43) expression, or growth in vitro of non-transformed (C10) or neoplastic (E9 and 82-132) murine lung epithelial cells. beta-Carotene enhanced GJIC and Cx43 expression and reduced the growth of C3H10T1/2 murine fibroblasts, however. These data indicate that the effects of beta-carotene on GJIC and growth are cell-specific which may partly explain why the carotenoid is an ineffective lung cancer chemopreventive agent.

Animals↗

PubMed1996

Gap-junction disassembly and connexin 43 dephosphorylation induced by 18 beta-glycyrrhetinic acid.

Gap-junction channels connect the interiors of adjacent cells and can be arranged into aggregates or plaques consisting of hundreds to thousands of channel particles. The mechanism of channel aggregation into plaques and whether plaques can disaggregate are not known. Many carcinogenic and tumor-promoting chemicals have been identified that inhibit cell-cell gap-junctional coupling. Here, we provide morphological evidence that 18 beta-glycyrrhetinic acid (18 beta-GA), a saponin isolated from licorice root that is an inhibitor of gap-junctional communication, caused the disassembly of gap-junction plaques in WB-F344 rat liver epithelial cells. This effect was dose (5-40 microM) and time dependent (1-4 h treatment). Gap-junction channels in WB-F344 cells are comprised of connexin 43 (Cx43), and the protein is phosphorylated to a species known as Cx43-P2 coincident with the assembly of channels into plaques. Consistent with this, the disassembly of plaques induced by 18 beta-GA was correlated with decreases in Cx43-P2 levels and increases in nonphosphorylated Cx43. Biochemical evidence indicated that these changes in the P2 and NP forms of Cx43 represented 18 beta-GA-induced dephosphorylation of Cx43-P2 and not its degradation or the inhibition of Cx43-NP phosphorylation. Okadaic acid and calyculin A, which are inhibitors of type 1 and type 2A protein phosphatases, prevented the dephosphorylation of Cx43, suggesting that one or both of these phosphatases were involved in Cx43 dephosphorylation. These data indicate that 18 beta-GA causes type 1 or type 2A protein phosphatase-mediated Cx43 dephosphorylation coincident with the disassembly of gap-junction plaques.

Animals↗

PubMed1996

Localization and function of the connexin 43 gap-junction protein in normal and various oncogene-expressing rat liver epithelial cells.

Clones of rat liver epithelial cells genotypically altered by mutation or by a variety of oncogenes were analyzed by microinjection-dye transfer, immunofluorescence confocal microscopy, and western blotting to determine at what level and to what degree these transformations disrupted gap-junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43). Compared with normal rat liver epithelial cells, cells neoplastically transformed by src, neu, ras, and myc/ras all displayed reduced degrees of GJIC, reduced levels of membrane-associated Cx43 plaques, and hypophosphorylation of Cx43. Confocal analysis further demonstrated that the Cx43 protein was localized, at least in part, to the nucleus rather than to the plasma membrane in the src- and neu-transformed cells, but not in the ras- and myc/ras-transformed cells. Nuclei isolated from WB-neu cells showed substantially higher levels of Cx43 on western blotting than did nuclei from WB-neo control cells, supporting the idea that the nuclear-localized immunopositive material detected by confocal microscopy was Cx43 protein. In a GJIC-deficient mutant rat liver epithelial cell line containing normal numbers of plasma membrane-localized Cx43 plaques that appeared to be reduced in size, the Cx43 protein was also found to be hypophosphorylated. Cells overexpressing myc, on the other hand, displayed a normal degree of GJIC, increased levels of plasma membrane-localized Cx43 plaques, and hyperphosphorylation of the Cx43 protein. Cells expressing raf, previously shown to be GJIC competent, showed Cx43 immunostaining patterns similar to those in normal cells, whereas a cell line established from a tumor induced by injection of these raf-expressing cells into a mouse showed a marked reduction in GJIC and plasma membrane-associated Cx43 immunostaining. These data suggest that altered localization of the gap-junction protein Cx43, mediated in part by changes in the phosphorylation of this protein, contributes to the disruption of GJIC in neoplastically transformed rat liver epithelial cells.

Animals↗

PubMed1996

Inhibition of gap junctional intercellular communication by barbiturates in long-term primary cultured rat hepatocytes is correlated with liver tumour promoting activity.

Rodent liver tumor formation can be promoted by certain barbiturates and this may involve their ability to inhibit hepatocyte gap junctional intercellular communication (GJIC). In order to address the mechanisms and specificity of action of barbiturates on hepatocyte gap junctions, we have compared the effects of liver tumor-promoting barbiturates (phenobarbital, sodium barbital and amobarbital: PB, SB and AB, respectively) and a non-liver tumor-promoting barbiturate (barbituric acid: BA) on primary cultured rat hepatocyte GJIC and connexin32 (Cx32) expression after short (1-24 h) and long (2-14 days) treatment. GJIC was evaluated by fluorescent dye microinjection (dye-coupling); Cx32 expression was monitored by Northern blot, Western blot and immunohistochemistry. Both parameters were maintained at high levels over 14 days by coculture of the cells with WB-F344 rat liver epithelial cells in the presence of dexamethasone. Treatment with PB (2 mM) for 1 h sharply reduced dye-coupling from approximately 90-30%, but the cells fully recovered by 24 h. No inhibition was seen with the other barbiturates over this 1-day treatment period. Longer treatments (2-14 days) with the promoters PB, SB and AB, however, gradually reduced hepatocyte dye-coupling to approximately 30-50%. The non-promoter, BA, did not affect hepatocyte GJIC. These decreases in hepatocyte dye-coupling occurred without changes in Cx32 or gap junction expression. Dye-coupling of WB-F344 cells and expression of their predominant gap junction protein, connexin43 (Cx43), were also not affected. Thus, the inhibition of GJIC was specific to liver tumor promoting barbiturates in hepatocytes, was time-dependent and was not due to altered Cx32 expression.

Animals↗

PubMed1996

Gap junction endocytosis and lysosomal degradation of connexin43-P2 in WB-F344 rat liver epithelial cells treated with DDT and lindane.

Treatment of WB-F344 rat liver epithelial cells with DDT (1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane) or lindane induces a loss of gap junction plaques and a decrease in the phosphorylated gap junction protein connexin43-P2 (Cx43-P2), which is associated with the plaques. In this study we have considered several mechanisms. The loss of junctional plaques could be due to disaggregation of junctional particles or to endocytosis of the plaques, while the loss of Cx43-P2 could be due to dephosphorylation or degradation. Immunohistochemical analyses of DDT- or lindane-treated cells revealed a reduction in plasma membranous Cx43-positive gap junction plaques coincident with the appearance of Cx43-positive punctate cytoplasmic structures. The cytoplasmic Cx43-positive structures eventually disappeared after 4 h treatment. Diffuse Cx43-positive plasma membranous staining was not seen following DDT or lindane treatment. Western blot analyses of these cells indicated that Cx43-P2 decreased in a time-dependent manner that paralleled the disappearance of gap junction plaques from the plasma membrane. The loss of Cx43-P2 was not due to dephosphorylation, since no increase in non-phosphorylated (Cx43-NP) or other phosphorylated (Cx43-P1) forms of the protein were evident. The decrease in Cx43-P2 and the disappearance of cytoplasmic Cx43-positive structures were prevented by colchicine and chloroquine, which suggests that Cx43-P2-containing plaques were internalized and degraded in lysosomes. In addition, two small (approximately 18 and approximately 22 kDa) bands appeared in Western blots coincident with the loss of Cx43-P2 and may be degradation products of the protein. These immunohistochemical and biochemical data strongly suggest that the loss of gap junction plaques and of Cx43-P2 in WB-F344 cells treated with DDT and lindane were due to endocytosis of the plaques and degradation of Cx43-P2 in lysosomes.

Animals↗

PubMed1996

Regulation of rat Na+/Pi cotransporter-1 gene expression: the roles of glucose and insulin.

Cytosolic inorganic phosphate (P(i)) is important for glucose metabolism. It plays a role in homeostatic regulation of glucose by insulin and glucagon. Recently, we isolated two cDNA clones for rat Na+/P(i) cotransporter-1 (rNaPi-1) and demonstrated that they are expressed primarily in the rat liver and kidney. We now report that the expression of rNaPi-1 in these tissues is regulated by fasting and streptozotocin-induced diabetes. Using rat hepatocytes in primary culture, we also demonstrate that glucose and insulin upregulate rNaPi-1 expression, whereas glucagon and elevated intracellular adenosine 3',5'-cyclic monophosphate levels downregulate its expression. Because 2-deoxyglucose exhibits no effect on rNaPi-1 gene expression, we suggest that some metabolite accumulated during glucose metabolism may be responsible for the effects of glucose and insulin on rNaPi-1 gene expression. Our data also reveal that other known Na+/P(i) cotransporter genes, NaPi-2 and Ram-1 (a receptor for amphotropic murine retrovirus), are not regulated by insulin and glucose. It is therefore proposed that various subtypes of Na+/P(i) cotransporters are differentially regulated and that each subtype may be involved in a specific cellular function, rNaPi-1 may be responsible for Pi uptake by liver and kidney for glucose metabolism, whereas NaPi-2 may play a key role in P(i) reabsorption in the kidney.

Animals↗

PubMed1995

Inhibition of gap junctional intercellular communication and enhancement of growth in BALB/c 3T3 cells treated with connexin43 antisense oligonucleotides.

Many studies have correlated reductions in gap junctional intercellular communication (GJIC) with altered cellular growth, tumor promotion, and neoplastic transformation. To test directly whether reduced GJIC affects cellular growth, GJIC was inhibited in murine BALB/c 3T3 fibroblasts by treatment with a phosphorothioate-modified antisense oligonucleotide targeted against the connexin43 translation start codon, and in vitro cell growth was monitored. The cells were incubated with the oligonucleotide (0.1-0.5 microM) in liposomes in serumless culture medium for 16 h; washed and refed with serum-containing medium; and analyzed for dye-coupling, connexin43 protein and mRNA levels, and cell growth over the next 5 d. The antisense oligonucleotide inhibited dye-coupling and reduced connexin43 protein levels in a concentration-dependent manner but had no effect on connexin43 mRNA levels. Cell growth rate was not affected, but saturation density was increased approximately threefold by the oligonucleotide. These data support a role for GJIC in the establishment of contact inhibition of in vitro cell growth.

3T3 Cells↗

PubMed1995

Changes in gap junction permeability, gap junction number, and connexin43 expression in lindane-treated rat liver epithelial cells.

The pesticide lindane (gamma-hexachlorocyclohexane) is a mammalian neurotoxin and hepatocarcinogen. Lindane can inhibit gap junctional intercellular communication (GJIC) and this effect may contribute to its toxic properties. The mechanism of inhibition of GJIC by lindane in WB-F344 rat liver epithelial cells was studied to determine if altered gap junction permeability, gap junction number, and/or gap junction protein (connexin43) expression were involved. GJIC was monitored by fluorescent Lucifer Yellow CH dye microinjection (dye-coupling). Gap junction number was quantified visually after indirect immunostaining of gap junctions using an anti-connexin43 monoclonal antibody. Connexin43 mRNA and protein levels were determined by Northern and Western blotting, respectively. Short-term treatment (10-30 min) with lindane (50 microM) resulted in the rapid (within 10 min), nearly complete loss of dye-coupling but no changes in gap junction number of connexin43 mRNA or protein levels. Medium-term treatment (1-4 hr) resulted in the loss of dye-coupling, gap junctions, and phosphorylated connexin43 proteins. Long-term treatment (1-14 days) led to reductions in dye-coupling, total connexin43 protein, and connexin43 mRNA. Nuclear run-on assays indicated that transcription of the connexin43 gene was reduced nonspecifically by lindane. These data indicate that reductions in gap junction permeability, number, and expression may be involved in the inhibition of GJIC depending on pesticide treatment duration.

Animals↗

PubMed1995

Down-regulation by butylated hydroxytoluene of the number and function of gap junctions in epithelial cell lines derived from mouse lung and rat liver.

The mouse pneumotoxicant and lung and liver tumor promoter butylated hydroxytoluene (BHT) was examined for its effects on gap junctional intercellular communication (GJIC) in mouse lung epithelial (C10) and rat liver epithelial (WB-F344) cell lines. GJIC, as measured by fluorescent dye microinjection, was inhibited in both types of cells by BHT in dose- and time-dependent fashions. Inhibition was detected in WB-F344 cells at BHT concentrations > or = 62.5 microM and in C10 cells at concentrations > or = 150 microM after 4 h treatment. Inhibition occurred within 15-30 min and was reversed by removing BHT from the culture medium. The highly toxic BHT metabolite 6-t-butyl-2-(hydroxy-t-butyl)-4-methylphenol (BHTOH) and the non-toxic BHT metabolite, 2,6-di-t-butyl-4-hydroxymethylphenol (BHTBzOH) were also tested. In both cell lines BHTOH was a more potent inhibitor of GJIC than BHT, whereas BHTBzOH was ineffective. The mechanisms of inhibition of GJIC by BHT were also examined. The initial rapid inhibition detected within 15-30 min may have been due to gap junction channel closure or blockage, since no changes in gap junction number, connexin (Cx) 43 levels or Cx43 phosphorylation were observed. By 2-4 h, however, gap junctions were internalized into the cytoplasm, the number of immunodetectable plasma membrane gap junctions was reduced and phosphorylated Cx43-P2 was decreased. Treatment of the cells for 24 h with 12-O-tetradecanoylphorbol-13-acetate (TPA) prevented inhibition of GJIC by TPA, but not by BHT. Western blot analyses of TPA-treated WB-F344 or C10 cells revealed the presence of a hyperphosphorylated form of Cx43 (Cx43-P3) and no reduction in Cx43-P2, in contrast to BHT-treated cells. These data suggest that BHT and TPA inhibit lung and liver epithelial cell GJIC through distinct mechanisms.

Animals↗

PubMed1995

In vitro growth inhibition of neoplastically transformed cells by non-transformed cells: requirement for gap junctional intercellular communication.

We examined whether the inhibition of neoplastically transformed cell growth by co-cultured non-transformed cells involved gap junctional intercellular communication (GJIC). The growth of poorly communicating (approximately 25-35% dye-coupled cells), Ha-ras and neu oncogene-transformed WB-F344 rat liver epithelial cells was inhibited by co-culture with highly communicating (90-95% dye-coupling), non-transformed WB-F344 cells. Inhibition was dependent upon heterologous cell-cell contact and required that the non-transformed cells were GJIC competent. GJIC-deficient mutant WB-F344 cells did not suppress transformed cell growth. Restoration of mutant cell GJIC by transfection with rat connexin43 cDNA restored growth-inhibiting activity. These results clearly demonstrate a role for GJIC in the inhibition of transformed cell growth by non-transformed cells.

Animals↗

PubMed1994

Changes in gap-junction permeability, phosphorylation, and number mediated by phorbol ester and non-phorbol-ester tumor promoters in rat liver epithelial cells.

The effects of three tumor promoters on gap-junction permeability; connexin 43 and 26 mRNA levels, protein levels, and phosphorylation; and the numbers of gap-junctional membrane plaques were studied in the rat liver epithelial cell line WB-F344 to determine whether changes in these parameters correlated with the inhibition of gap-junction function. 12-O-tetradecanoylphorbol-13-acetate (TPA; 10 ng/mL), dieldrin (10 micrograms/mL), and heptachlor epoxide (10 micrograms/mL) inhibited gap-junctional intercellular communication (GJIC) assayed by fluorescent dye transfer by 80-90% after a 5-min exposure and by more than 90% within 1 h. Decreases in steady-state connexin 43 mRNA levels were detected by northern blot analysis within 1 h and paralleled changes in steady-state beta-actin mRNA, but these changes did not occur rapidly enough to account for the rapid loss of gap-junction function. A substantial loss in the number of connexin 43 immunostained gap-junctional membrane plaques was detected after a 15-min exposure to all three promoters, but little change had occurred at 5 min. Western blot analyses using connexin 43-specific antibodies showed changes in the degree of connexin 43 phosphorylation for all three tumor promoters. TPA induced the appearance of a fourth connexin 43-immunoreactive band (P3) and a concomitant decrease in the relative intensity of the unphosphorylated (P0) band within 5 min of treatment. P3, in addition to bands P1 and P2, disappeared after treatment with alkaline phosphatase. In contrast, dieldrin and heptachlor expoxide induced loss of P2 with a concomitant increase in the relative staining intensity of P0 within 1 h of exposure, but no changes were seen after 5 min. Connexin 43 phosphorylation levels recovered in parallel with the recovery of GJIC for all three tumor promoters. Connexin 26 mRNA levels showed little change after a 1-h exposure to three promoters, but reductions in connexin 26 immunofluorescent staining were observed. These results suggest that (i) TPA-induced hyperphosphorylation of connexin 43 occurred fast enough to account for inhibition of GJIC, (ii) dieldrin and heptachlor expoxide modulated connexin phosphorylation in a manner different from TPA by promoting hypophosphorylation of connexin 43, (iii) redistribution of plasma membrane gap-junctional plaques after treatment with phorbol ester and non-phorbol-ester tumor promoters occurred subsequent to changes in gap-junction permeability, and (iv) changes in connexin mRNA levels could not account for the losses in fluorescent dye coupling induced by these promoters.

Animals↗

PubMed1994

Loss of gap junctions from DDT-treated rat liver epithelial cells.

The mechanism by which the liver tumor promoter 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) inhibits gap junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells could involve gap junction loss and/or decreased gap junction channel permeability. We examined these two possibilities in the present study. Immunohistochemical studies using antibodies specific to connexin43, the major gap junction protein expressed by these cells, revealed that gap junction number and size were reduced during exposure to DDT. The reductions in gap junctions (33-91%) correlated with dose-dependent (1-10 microM) and time-dependent (0.5-4 h) decreases in cell-to-cell fluorescent dye-coupling (64-85%), as well as cellular levels of phosphorylated connexin43. These effects were reversible following removal of the tumor promoter from the culture medium, although cycloheximide reduced the level of gap junction reformation. The losses in gap junctions were not due to decreased connexin43 gene expression since steady-state levels of connexin43 mRNA were not similarly affected by DDT. Fenarimol (10 microM), a structural analog of DDT, did not inhibit GJIC and had no effect on gap junction structure or connexin43 expression. These data suggest that the inhibition of GJIC by DDT resulted from the removal of gap junctions from the plasma membrane and their degradation rather than simply a decrease in their permeability.

Animals↗

PubMed1994

Growth inhibition of rat liver epithelial tumor cells by monoterpenes does not involve Ras plasma membrane association.

The role of altered ras oncoprotein (Ras) farnesylation and membrane association in the growth inhibitory effects of several monoterpenes (limonene, perillic acid, perillyl alcohol, menthol, pinene and cineole) was investigated in rat liver epithelial cells. All of the above compounds except cineole inhibited the growth of viral Ha-ras-transformed rat liver epithelial cells (WB-ras cells) at concentrations of 0.25-2.5 mM. These cells, however, were not necessarily more sensitive to these compounds compared to non-transformed and viral raf-transformed rat liver epithelial cells. Growth inhibition by limonene, perillic acid and pinene was only partially restored (20-50%) by supplementing the culture medium with 2 mM mevalonic acid. Western blot analyses of cytosolic and membranous fractions of WB-ras cells treated with monoterpenes indicated no change in Ras distribution. In contrast, lovastatin, a potent inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and Ras farnesylation, specifically reduced WB-ras cell growth and increased cytosolic levels of Ras. Thus, monoterpene-induced growth inhibition of rat liver epithelial cells was dissimilar to lovastatin and did not appear to involve altered Ras plasma membrane association.

Animals↗

PubMed1994

Enhancement of liver cell gap junction protein expression by glucocorticoids.

Gap junctional intercellular communication (GJIC) and the expression of gap junction proteins (connexins) may be involved in growth regulation and neoplastic transformation. The mechanisms of connexin gene regulation in normal and neoplastic tissues are poorly understood. In this study, the glucocorticoids, dexamethasone and hydrocortisone, enhanced fluorescent dye-coupling in primary cultured rat hepatocytes and MH1C1 rat hepatoma cells. Other types of steroids (beta-estradiol, testosterone, aldosterone and progesterone) had no effect. Northern blot, Western blot, nuclear run-on and immunohistochemical analyses showed that glucocorticoids enhanced the expression of connexin32 in these cells in a dose- and time-dependent fashion. Connexin26 expression was also enhanced slightly by dexamethasone in hepatocytes, but not MH1C1 cells. Connexin43 expression in these cells was not affected by steroids. In WB-F344 rat liver epithelial cells, which were highly coupled and expressed high levels of connexin43 and no detectable connexin32 or connexin26, dexamethasone had no effect on coupling or connexin expression. These results indicate that dye-coupling and the expression of connexin32 and connexin26, but not connexin43, were upregulated by glucocorticoids in a cell-specific manner. These effects on GJIC and connexin expression may be involved in the induction of hepatic differentiation and inhibition of growth.

Animals↗

PubMed1993

Specific nuclear protein binding to a negative regulatory element on the human CYP1A1 gene.

Employing reporter gene/CYP1A1 chimeric plasmids, we previously identified a 275-base pair (bp) cis-element (-833 to -558; NRE275) that down-regulated the CYP1A1 promoter. In the present study, this negative regulatory activity was further localized to two subfragments of 105 bp (-833 to -728; NRE105) and 170 bp (-728 to -558; NRE170), each of which exhibited activity with a heterologous promoter/enhancer. Co-transfection studies demonstrated a dependence on cellular trans-acting factors present at limiting concentrations. Electrophoretic mobility shift assays revealed the presence of protein(s) that specifically bound to NRE275, NRE105, and NRE170. Based upon competition studies, the protein(s) that bound to NRE105 appeared to recognize sites similar to those recognized by the NRE170-binding proteins. DNase I footprint analysis of NRE105 demonstrated nuclear protein binding to a 21-bp palindrome (-794 to -774). Protection was also observed along conserved guanine/cytosine-rich sequences that flank the palindrome, but in a strand-specific manner. Guanine residues involved in protein binding were identified by methylation interference experiments. Based on transient expression assays with each of the three NRE105 components, all three appear to be necessary for complete negative regulatory activity. However, it is clear the palindrome is the most important sequence with the guanine/cytosine-rich elements playing an ancillary role.

Base Sequence↗

PubMed1993

Gap junctional intercellular communication in mouse lung epithelial cell lines: effects of cell transformation and tumor promoters.

Gap junctional intercellular communication (GJIC) is reduced by neoplastic transformation and treatment with tumor promoters in many types of cells but few data exist for the lung. GJIC was therefore evaluated in non-transformed (C10) and transformed (E9, 82-132, and PCC4) mouse lung epithelial cell lines and in C10 cells treated with tumor promoters. GJIC was assessed by fluorescent dye microinjection (dye-coupling). Dye-coupling levels were highest in C10 cells (85-90% communicating cells) followed by 82-132 cells (40-50%), E9 cells (15-20%), and PCC4 cells (3-10%). Indirect immunofluorescent staining with anti-gap junction protein (connexin) antibodies revealed that C10 cells expressed gap junctions comprised of connexin43, but not connexin32 or connexin26. The tumor promoters, butylated hydroxytoluene (BHT), 12-O-tetradecanoylphorbol-13-acetate (TPA), and p,p'-dichlorodiphenyltrichloroethane (DDT), inhibited dye-coupling in C10 cells but phenobarbital (PB) did not. BHT promotes mouse lung tumor formation, PB does not, while the effects of TPA and DDT on lung tumor development have not been reported. These data indicate that cell transformation and certain tumor promoters reduce GJIC in mouse lung epithelial cells and demonstrate correlations between the in vitro inhibition of GJIC and lung tumor promotion.

Animals↗

PubMed1993

Enhancement of gap junctional intercellular communication in tumor promoter-treated cells by components of green tea.

Green tea (Camellia sinensis) has been reported to inhibit tumor promotion in vivo and in vitro. Many tumor promoters inhibit gap junctional intercellular communication (GJIC) which may be an important mechanism of promotion. In the present study, we hypothesized that green tea would enhance GJIC in promoter-treated cells. An aqueous extract of green tea (GTE) and several of its constituents were tested for their effects on GJIC in p,p'-dichlorodiphenyltrichloroethane (DDT)-, 12-O-tetradecanoylphorbol-13-acetate (TPA)- and dieldrin-treated WB-F344 rat liver epithelial cells. All three promoters inhibited GJIC in a dose-responsive manner at non-cytolethal concentrations. (GTE (10-80 gamma/ml) enhanced GJIC 20-80% in promoter-treated cells. (-)-Epigallocatechin gallate and (-)-epicatechin gallate also enhanced GJIC in DDT-treated cells, but no effects were seen with (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, caffeine, or theobromine. These data suggest GTE may inhibit tumor promotion by enhancing GJIC and that the most active components are the catechin gallates.

Animals↗